Process for preparing alpha amylase

ABSTRACT

ALPHA AMYLASE CAN BE PRODUCED BY FERMENTING, UNDER AEROBIC CONDITIONS, NUTRIENT MEDIA CONTAINING AT LEAST A PORTION OF FOWL EXCREMENT. THE FERMENTATION IS ACCOMPLISHED BY MEANS OF MICROORGANISMS SELECTED FROM THE GROUP CONSISTING OF THOSE PSEUDOMONAS, CORYNEBACTERIA, CELLUMONAS, BACILLUS, ASPERGILLUS AND CANDIDA CAPABLE OF PRODUCING ALPHA AMYLASE BY METABOLIZING FOWL EXCREMENT AS A SOURCE OF ASSIMILABLE CARBON AND/OR A SOURCE OF ASSIMILABLE NITROGEN. PARTICULARLY GOOD PRODUCTION IS OBTAINED BY USING FIVE NEWLY DISCOVERED STRAINS, BACILLUS CEREUS A.T.C.C. NO. 21768, BACILLUS CEREUS A.T.C.C. NO. 21769, BACILLUS CEREUS A.T.C.C. NO. 2170, BACILLUS CEREUS A.T.C.C. NO. 21771, BACILLUS CEREUS A.T.C.C. NO. 21772. WHEAT CHAFF IS AN EXCEPTIONALLY USEFUL NUTRIENT INGREDIENT AND GLYCINE IS A PARTICULARLY EFFECTIVE STIMULANT FOR SOME OF THESE PROCESSES.

`April 30, 1974 J. D, DOUROS, JR.. ETAL 3,808,102

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PROCESS FOR PREPARING ALPHA AMYLASE Filed May 5. 1972 7 sheets-sheet e EMZYME KINETICS CURVE FOR CRUDE `BACILLUS CEREUS ATCC 2l768 AMYLASE ASSAYED AT pH 6.9; 25C; VARIABLE INCUBATION TIMES 40o 4 l I o, 30o O LU DE 20o LLI l X IOG O 2 4 6 8 IO I2 I4 I6 I8 2O INCUBATION TIME IN MINUTES F/GURE 6 EFFECT OF pH ONCRUDE BACILLUS CEREUS ATCC 2|768 AMYLASE ACTIVITY ASSAYED FOR 3 MIN INCUBATION TIME; 25C; VARIABLE pH O 4 6' 8 IO l2 F/GURE 7 I April 30,1974 .1.0. DouRosv. JR., ETAL 3,808,102

PROCESS FOR PREPARING ALPHA AMYLASE Filed May 5, 19712 Sheets-Sheet Cv' A EFFECT OF TEMPERATURE ON CRUDE` BACILLUS CEREUS ATCC 2I768 -AMYLASE STABILITY I pH 6.9

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O x l v| Ol I 2 3 TIME INCUBATION AT 70C IN HOURS F/GURE 8 v EFFECT OF TEMPERATURE lON CRUDE BACILLUS CEREUS ATCC 2I768 AMYLASE ACTIVITY ASSAYED AT pH 6.9; 3 MIN. INCUBATION; VARIABLE TEMPERATURES ZOO Lu IOO IL l x TEMPERATURE IN c United States Patent O U.S. Cl. 195-65 22 Claims ABSTRACT OF THE DISCLOSURE Alpha amylase can be produced by fermenting, under aerobic conditions, nutrient media containing at least a portion of fowl excrement. The fermentation is accomplished by means of microorganisms selected from the group consisting of those Pseudomonas, Corynebacteria, Cellumonas, Bacillus, Aspergillus and Candida capable of producing alpha amylase by metabolizing fowl excrement as a source o f assimilable carbon and/ or a source of assimilable nitrogen. Particularly good production is obtained by using five newly discovered strains, Bacillus cereus A.T.C.C. No. 21768, Bacillus cereus A.T.C.C. No. 21769, Bacillus cereus A.T.C.C. No. 21770, Bacillus cereus A.T.C.C. No. 21771, Bacillus cereus A.T.C.C. No. 21772. Wheat chaff is an exceptionally useful nutrient ingredient and glycine is a particularly elective stimulant for some of these processes.

BACKGROUND OF THE INVENTION Processes for producing alpha amylase by microorganism fermentation are well known. Typically, nutrient media containing various sources of starch, protein and minor nutrients are formed, sterilized, cooled and inoculated with cultures of various bacteria, yeasts or molds which are capable of producing alpha amylase by metabolizing the nutrient media employed in the particular process. Developments in this art usually involve finding improved nutrient media and growing conditions for particular alpha amplase producing microorganisms. Alpha amylases produced by such processes are noted for their ability to hydrolyze starch to give sugar products such as dextrin, glucose, maltose etc. and are used in numerous commercial, industrial, pharmaceutical and scientific applications. Among these uses may be mentioned, food and beverage preparation, adhesive material manufacture, pollution oontrol, digestive aids, disease treatments and aids in basic research such as that concerned with elucidating the structures of various complex carbohydrates. Representative nutrient media, microorganisms, fermentation processes and uses for alpha amylase are found in United States patents: 2,695,863; 3,012,944; and 3,414,479.

SUMMARY OF THE INVENTION One aspect of our invention involves using newly discovered Bacillus strains to produce alpha amylase. These strains can be cultured from fowl excrement by methods hereinafter described and have been given the designations: Bacillus cereus A.T.C.C. No. 21768, Bacillus cereus A.T.C.C. N0. 21769, Bacillus cereus A.T.C.C. No. 21770, Bacillus cereus A.T.C.C. No. 21771, and Bacillus cereus A.T.C.C. No. 21772.

Another important aspect of this invention is our discovery that alpha amylase production by microorganisms ICC selected from the group consisting of Pseudomonas, Corynebacteria, Aspergillus, Cellumonas, Bacillus, or Candida on nutrient media containing conventional sources of starch, protein and minor nutrients hereinafter described can be greatly enhanced when at least a portion of the nutrient media is fowl excrement. Particularly good alpha amylase production in conventional nutrient media containing a portion of fowl excrement is obtained by using the aforementioned Bacillus strains, Bacillus cereus A.T.C.C. Numbers 21768 through 21772. A complete morphological description of each of these microorganisms will be Vgiven in subsequent sections of this application. This invention also describes how alpha amylase can be produced by using fowl excrement as the sole source of assimilable carbon and nitrogen. Still another aspect of this invention is to provide an improved nutrient medium for culturing those microorganismscapable of producing ferment mixtures rich in alpha amylase. To this end the applicants have discovered that wheat chaff provides exceptional nutrient qualities, particularly for the Bacillus cereus No. 21768 strain used in this invention. The applicants have also discovered that glycine stimulates alpha amylase production by the new strains of this invention in a nutrient media which also contains wheat chaff.

The above fermentation processes result in culture fluids which are readily processed by conventional precipitation, filtration and exchange steps to recover alpha amylase. Precipitation usually is accomplished by the addition of a salt of a divalent cation to coagulate the alpha amylase. After precipitation, the alpha amylase produced by the methods of our invention is most conveniently collected by using exchange chromatography of DEAE Cellulose and KCL or other elution solvents.

A more complete understanding of the nature and scope of this invention can be made by referring to the following detailed descriptions, wherein presently preferred illustrative embodiments of the invention are set forth. These descriptions, particularly the examples are given for purposes of illustration and should not be considered as limitations on the scope of this in'vention.

EXAMPLE l Assay method used to establish invention The preferred standardized technique of alpha amylitic assay used to establish this invention is accomplished according to the general methods of Fischer and Stein (Biochemical Preparation, v.7, 1960) wherein alpha amylase activity is measured by the use of dinitrosalicylic acid (DNS).

The substrate used in this technique is a 1% solution of soluble starch (Noredux Standard, B. Siegfried and Company, Zofingen, Switzerland) dissolved in boiling water and made up with a 0.02 M sodium glycerophosphate-HCl buffered to pH 6.9. The stopping reagent is an alkaline solution of dinitrosalicylic acid prepared by suspending 20 gm. of 3,5-dinitrosalicylic acid in 400 ml. of water. A solution of 32 gm. of NaOH in 800 ml. of water is added dropwise under efficient stirring, and, if necessary, gentle heat in a water bath until a clear solution is obtained. The rest of the NaOH solution is then added and 600 gm. of potassium sodium tartrate is added in small portions. Water is added to a final volume of 2 liters. The solution is then filtered through a large coarse sin- 3 tered-glass filter and stored at room temperature in the dark. i

The actual alpha amylase assays proceed as follows. One ml. of the substrate solution is added to l ml. of

each of the test alpha amylase solutions diluted in 0.002 M sodium glycerophosphate-HCI buffer at the same pH as the substrate solution. After exactly 3 minutes of incubation at 25 C., 2.0 ml. of the stopping reagent are A.T.C.C. number 21768 21769 21770 21771 21772 Source Turkey manure, Chicken manure, Chicken manure, Chicken manure, Chicken manure, chicken manure. turkey manure. turkey manure. turkey manure. turkey manure. Media Dextrose starch agar. Dextrose starch agar. Dextrose starch agar. Dextrose starch agar- Dextrose starch agar. Growth temp.. 37 C 3 37 37 37 C. Growth nH 6.8. 6.82 6.8 6.8- 6.8. Gram strain Gram 3 microns.-." Gram lVv-Zp.- Gramul+el3ii chains, Gram 195i; Gram 3a.

V86 a Spore Eccentric l, 1.5 it Eccentric -l, 2p Eccentric 1.5;4 Eccentric .75p Eccentric 1.5 Peptone iron i Starch hydrolysis.. -I- Litmus milk l day reduction, 1 day alkaline 1 day hydrolysis 1 day hydrolysis 1 day hydrolysis days). days). (5 days (5 days). days). Colony morphology..- White mucoid, Grey white mucold-.- White mucoid, spreading White mucoid, White mucoid,

glistening. dark with age. glistening. spreading. Indole- Voges Proskauer Nitrate rndiirfinn -NO EAS Methyl red. Kosercitrate-- Motiiity. -i- -l- -l- Cassia@ -I- Gelatin Stat- -i- N ora-See the following table:

Bucal," cmu, A Taa Nm 21768 added, thetube is placed in boiling water for 5 minutes and then cooled, and the reaction mixture is diiuted with Carbohydrate data Acid Growth Gas 20 ml. of water. The color is read in a Kle1t-Summerson Photoelectric Colorimeter equipped with a green iilter mm1 No. 54 (spectral range SOO-570 mit). The extinction Celiobioseiannimi -it 30 value is converted to milligrams of maltose from a standffifglf I i and curve established with D (-l-)-maltose hydrate. No milin starch or buffer is added to this maltose solution. The apgms ghtm plicants have found that the best results are obtained cin-- when 1.0i 0.5 mg. of maltose are produced during the $2,211,201' l 35 reaction. This corresponds to the use of 0.3 to 1.0 ,.tgm. sucres of alpha amylase in the assay system. The applicants have PTE? T. defined an alpha amylase activity unit (DNS Unit) as that amount of enzyme that produces 1 mg. of maltose under the above conditions. Unless otherwise specied the re- BWM uw ATCC Niv-21769 40 suits of the tests for alpha amylase found with the follow- Imv'm ing examples are expressed in DNS'Units.

ni L1 finden EXAMPLE 2 sffiliffni I I Selection, screening and identification techniques ihn Giyrgni The isolation, selection and identification of the new rudnstrains Bacillus cereus A.T.C.C. Numbers 21768 through nnnitnl mitm.. 21772 may be accomplished according to known prini" ciples. OneA method used by the applicants is conducted Dextrns -iiai-fm 50 1n the following manner. Fusions of chicken excrement Mmm-5" sized to 350 mesh or greater are screened for alpha amylase leads on plates prepared with dextrose lstarch agar using 8 milliliters per plate. After 24, 4S and 72 ma m AT'C'C N' 21770 hour intervals the plates are flooded with an iodine soluimqiini tion composed of 11 grains of iodine and 22 grams of t potassium iodide in 500 ml. distilled water. Positive @532mm l' amylase production lis indicated by a clear or yellow zone Duif-imi indicating starch degradation. The testing is conducted lgfflm, 2 using basic, neutral and acidic pH adjusted agar but it saiiciii-- 60 should be noted that the acid test plates need about a 2% mig i l increase inthe agar content for proper identification; This sucia@ -iprocedure is carried out three times in triplicate for re- Detm 'l' confirmation of activity (3 positive, 1 positi've -1 nega- Maunose-- tive --l positive, or 3 negative results). Obvious in strain improved colonies are selected and again tested on dex- Bacmuawm A.T QQ No- 21771 trose starch agar plates with phosphate buffer pHs of 4.5; 6.8; and 8.4. These strains are not inhibited by the Innsttn; high pH of 8.4 or the low of 4.5. The tive isolated leads giiigiigim S have been deposited with the American Type Culture sorbiioi- 70 Collection, Washington, D.C., and have been assigned llffd- I the designations, Bacillus cereus A.T.C.C. No. 2176s, Gllyi'ciernl Bacillus cereus A.T.C.C. No 21769, Bacillus cereus f t I A.T.C.C. No; 21770, Bacillus cereus A.T.C.C. No. 2177i, lslffeltcse Bacillus cereus A.T.C.C. No. 21772. Drt'fa :i: i' 75 The cultural and microscopic characteristics of these strains are as follows.

Bacillus cereus A.T.C.C. No. 21772 Acid Carbohydrate data Growth Gas EXAMPLE 3 Purification of alpha amylase The general procedure for purification of the alpha amylase produced by each of the live Bacillus cereus strains of this invention is summarized below.

tested for their capacity to produce alpha amylase by comparison with two other known amylase producing microorganisms, Aspergillus niger, A.T.C.C. No. 15475, and Bacillus amyloliquefaciens A.T.C.C. No. 23843.

A loop of the spores taken from the dextrose starch agar slants of each of the new microorganisms and the two known alpha amylase producing microorganisms are transferred to individual Erlenmeyer flasks which contain 150 ml. of either a Spizzen broth, a Wheat Bran, or a Bacillus amylase. About l to 5 ml., 16 hour vegetative inoculum is used with the pH adjusted to 6.8. The flasks are then placed in 50 C. water for 1S minutes, then cooled, and the contents are transferred quantitively to a 150 ml. flask. The solution is adjusted to pH 4.8 with 2.0 normal sodium hydroxide solution, transferred to a 500 ml. Erlenmeyer flask, and diluted to about 200 ml. The contents are then transferred to a Z50-ml. volumetric flask and diluted to volume. Two hundred ml. are then centrifuged at 2000 r.p.m. for 15 minutes, and the supernatant Activity/mg. lyophilized te 'al Purica- Percent Step Activity ma n tion recovery 1 1 liter amylase WBF culture--. 30 units per ml. T.A.= 0 100 30,000 units. 2---- Dialysis vs. ca. Ac; NaC1.-- 34 units/ml. T.A.= 113 3 Lynphnina 8.3 gnu-4 units/mg. 11o

T.A.=33,200. 4...- Redlssolved, 60% (NHLMSO 2.2 gnLa-funitS/mg. T.A.= 3. 5X 102 precipitate. 30,800.

5.--. Hydroxylapetite, vol.=480 ml. 45 units/ml., 480 m1.= I2

6 Dial d1 mu n vol' T'A' 21'600' 21o 10o uits/ 25X 7o s an o za on mgmu m ysi y p T.A.=21,000. g

The details of these procedures are as follows. One liter of the supernatant on which each microorganism was grown is assayed for amylase activity and found to have -30 units per ml. Each of these solutions is individually dialyzed for two days against 12 liters of 0.01 M calcium acetate, 0.1% sodium chloride buffer, with 4-5 changes of this buler during the dialysis. The dialyzed solutions are assayed and found to have between 1 and 45 units per ml. Each dialyzed solution is lyophilized and the dry material (about 4 to 8.3 grams) is suspended in 300 m1. of distilled water. Each solution is brought to 60% saturation and the proteins are allowed to precipitate overnight in the cold. Each suspension is centrifuged and the supernatants are found to contain no appreciable amylase activity and are discarded. The pellets are resuspended in 50 ml. of the calcium acetate-sodium chloride buffer. These solutions are found to have amylase activities of 400 to 600 units per ml. and are ready for further purification by column chromatography on hydroxylapetite absorption resin. However, the applicants advise that a small amount of each material (10 m1. with 90 units per ml.) be applied to the column first in order to check the operation of the system. For each test the column is rst eluted with 0.1 M sodium phosphate buffer at pH 7.0, which generally gives two U.V. 280 my. absorbing peaks which have been designated A and B in the elution profiles which follow. However, these peaks possess little if any amylase activity. Therefore, the columns should be eluted with 0.5 M sodium phosphate buffer at pH 7.0. This procedure results in the elution of a third protein peak C which possess about 400-600 units of the total 950 units of amylase which is initially applied to the column. The elution profile for the protein peaks of each of the new strains is shown in FIGS. l through 5.

EXAMPLE 4 Alpha amylase activity of new strains vs. known alpha amylase producing strains Spore isolates of each of the new microorganisms Bacillas cereus A.T.C.C. Numbers 21768, 21769, 21770, 21771, and 21772 produced by the techniques of Example 2 are liquir is decanted into a dry ask. Fifty ml. of this liquor is pipetted into a -ml. test tube, 5 ml. of 5.0 normal hydrochloric acid is added, the test tube is stoppered loosely and heated in a boiling water bath for 3 hours and then cooled in an ice bath. The contents of the tube is transferred to a ml. volumetric ask, and adjusted with 2.0 normal sodium hydroxide to a phenolphthalen end point. After dilution to volume, a reducing sugar value, calculated as dextrose, is determined on an aliquot of the iinal solution. Under these conditions the alpha amylase content of the Bacillus cereus A.T.C.C. 2,1768, 21769, 21770, 21771, and 21772 organisms cultures attained a maximum in about 20 hours while the amylase content of the two known cultures did not attain their maximum growths until about 40 hours. The amylase production of these tests expressed in DNS units are summarized as follows.

NOTE. See the following:

Spizzen broth Boil 200 g. peeled diced potatoes in one liter of tap H2O for l hour. Filter through cotton.2 Add 5.0 mg. MnSO4. Adjust pH to 6.8. Make-up volume to 1 liter with tap Wheat bran filtrate- 100 g. wheat bran (Hamico, Shorts, Red Dog) 300 cc. tap H2O. Autoelave, strain through cheesccloth add 200 ml. tap H10 and autoclave. amylase 1-1euclne= 10 ing/1.

Bacillus KzHPO4=l4 g. Na cltrate=.2H1O, l g. media Ammonium sulphate=2 g.

Magnesium sulph.=7HzO, .2 g. l liter H2O pH to 7 Autoclave separately-5 g. glucose 7 EXAMPLE 5 Alpha amylase kinetics FIG. 6 shows a kinetics curve for the new Bacillus cereus A.T.C.C. Number 21768 strain of this invention.

The kinetics curves for the other four new Bacillus cereus strains of this invention are almost identical with that of the 21768 strain. Therefore, an analysis of the curves show that the reactions of these strains is generally linear to the point where 2.0 amylase units are formed. At this point the reaction rate decreases considerably. Therefore, one must be sure to have the alpha amylase diluted to a concentration which places it on the linear portion of the reaction. Under our assay conditions the activity should be less than 2.0 units per ml.

EXAMPLE 6 Effect of pH on amylase activity 'Each enzyme is assayed at 25 C. for three minutes but at various pHs. Both the enzyme solution and the 1% starch solutions are prepared in a buler solution at the pH in which the alpha amylase is going to be assayed. The results for Bacillus cereus A.T.C.C. No. 21768 are shown on FIG. 7. Again the curves for the other four strains are almost identical. Examination of the pH curves show maximal activity at about pH 7.5. Generally there is still about 30-40% of the maximal activity present at a pH of EXAMPLE 7 |Etect of prolonged heat treatment on stability of amylase activity Each amylase solution is subjected to 70 C. for zero time, l hour, 2 hours, 3 hours etc. 'Ihe alpha amylase samples are then cooled and assayed at 25 C. The results of these tests for each strain are very similar and are shown in FIG. 8. There is no loss of activity after 1 hour at 70 C. After 2 hours there is about a 20% loss of alpha amylase activity and after 3 hours incubation there is about a 30% loss in enzyme activity.

EXAMPLE 8 4Elect of temperature on activity The alpha amylase produced by each of the live new strains of this invention are assayed as above but at various temperatures. The alpha amylase solutions and starch substrate solutions are pre-warmed for live minutes at the assay temperature before the reaction was started. The results f these tests are almost identical and are shown on the .graph in FIG. 9. Examination of these temperat-ure curves shows that there is an almost linear increase in amylase activity from 25 to 45 C. Generally from 45 to 70 C. there is only a slight increase in amylase activity, and at `80 C. there is generally a 10-30% decrease in alpha amylase activity.

EXAMPLE 9 Additional experiments are carried out wherein the alpha amylase activity of the enzymes produced yby the new strains of this invention is compared to a known commercial (Novo) alpha amylase producing enzyme.

TABLE 1.--COMPARISON OF NOVO AMYLASE AND /LASE PRODUCED BY Bacillus Census A.T.C.C. NUMBER Etect of temperature on stability:

l. Percent activity loss alter 15 min.

et 70 C 7 0 0 2. Percent activity loss alter 30 min.

a 0 78 3. Percent activity loss after 1 hr. at

7 94 0 95 4. Percent activity loss after 2 hrs. at 70 C 2. 6 Eect o high temperature and high pH on amylase stability: (2) (i) 1. Percent loss after 15 mln. at pH 8.5 0

2. Percent loss after 30 min. at pH 8.5" 3. Percent loss after 15 min. pH 9.5. 4. Percent loss after 30 min. pH 9.5. 5. Percent loss attet' 15 min. pH 10.5.-

l Since the enzyme Was very unstable at high temp. (70 C.), the combined effect oi high temperature and high pH was not performed.

2 Not performed for same reason as stated lor novo amylase.

TABLE 2.COMPARISON OF NOVO AMYLASE AND 2Alg/(ISQILASE PRODUCED BY Bacillus cereus A.T.C.C. NUMBER Novo Crude Purified amylase 21769 21769 Specific activity: DNS units per milligram enzyme 20 5 50 Optimum p 5. 5 5. 0 5. 5 Percent optimum activity atpH 2.5 0 45 33 pH 8.5 51 60 55 pH 9.5 5 33 24 pH 10.5 2. 5 8 0 Etect of temperature on activity:

1. Optimum temperature, C 45 60 60 2. Percent increase of activity at optimum over activity at 25 C 43 150 135 3. Percent optimum activity et 80 C. 60 70 52 Etect of temperature on stability:

1. Percent activity loss after 15 min.

at 7 10 8 2. Percent activity loss after 30 min.

at 70 C B0 22 40 3. Percent activity loss alter 1 hr. at

70 C 94 3G 70 4. Percent activity loss alter 2 hrs. at

lSince the enzyme was very unstable at high temp. (70 0.), the combined etlect of high temperature and high pil was not performed.

l Not performed for same reason as stated lor novo amylase.

JlE PRODUCED BY Bacillus cereus A.T.C.C. NUMBER Novo Crude Purified amylase 21770 21770 Specific activity: DNS units per milligram enzyme i0 72 Optimum pH 5 5 4. 5 4. 5 Percent optimum activity at-- pH 2.5 0 40 2G 61 45 30 10 5 35 .20 pH 10.5 2 5 10 5 Effect of temperature on activity:

1. Optimum temperature, C 45 50 50 2. Percent increase of activity at optimum over activity at C.. 43 85 90 3. Percent optimum activity at 80 C- 60 70 72 l Eiect of temperature on stability: a

1, Percent activity loss after 15 mn.

at 70 C 7 0 0 2. Percent activity loss after min.

80 0 75 3. Percent activity loss after 1 hr.

70 C 94 5 95 4. Peigciit activity loss after 2 hrs. at lo 2() Effect of high temperature and high pH on amylase stability (i) 1. Percent loss after 15 min. at pH 8. 5 2. Percent loss after 30 min. pH 8:5 3. Percent loss after 15 min. pH 9.5 4. Percent loss after 30 min. pH 9.5 5. Percent loss after 15 min. pH 10.5 6. Percent loss after 30 min. pH 10.5

1 Since the enzyme very unstable at high temp. (70 C.), the combined effect of high temperature and high pH was not' performed.

l Not performed for same reason as stated for novo amylase.

TABLE 4.-COMPARISON OF NOVO AMYLASE AND AMY- PRODUCED BY Bacillus cereus A.T.C.C. NUMBER Novo Crude Purified amylase 21771 21771 S ic activit :DNS units milli m DBC e Y Per Ela 20 20 85 5. 5 5. 5 5 5 Percent o timum activit atpn 2.15) y o 52 42 55 pH 8 5- 51 48 33 pH 9.5 5 30 20 pH 10.5 a 5 15 Effect of temperature on activity:

1. Optimum temperature, C 45 55 60 2. Percent increase o! activity at optimum over activity at 25 C 43 90 90 G0 3. Percent optimum activity at 80 C- 60 70 68 Effect of temperature on stability:

1. Percent activity loss after 15 min.

at C .L 7 0 5 2. Percent activity loss after 30 min.

at 80 10 82 3. Percent activity loss after 1 hr. at

94 25 98 65 4. Percent activity loss after 2 hrs. at 39 l Since the enzyme was very unstable at high temp. (70 C.), the com bincd eect of high temperature and high pli was not performed.

2 Not performed foi' same reason as stated lor novo amylase. 5

TABLE 5.-COMPARISON OF NOVO AMYLASE AND AMY- LASE PRODUCED BY Bacillus cereus A.T.C.C. Number 21772 Novo Crude Purified amylase 21772 21772 Specific activity: DNS units per milli- Eicct of temperature un activity:

1. Optimum temperature, C 45 G0 60 2. Percentincrease oi activity at optimum over activity at 25 C 3. Percent optimum activity at C- 60 69 62 Effect oi temperature on stability:

1. Percent activity loss after 15 min.

at C 7 5 8 2. Percent activity loss after 30 min.

at 70 C 80 8 16 3. Percent activity loss after 1 hr. at

70 C 94 22 4. Percent activity loss after 2 hrs. at 88 70 C 30 (2) Effect of high temperature and high pH 40 on amylase stability (l) (2) 1. Percent loss after l5 min. at pH 8.5 10 2. Percent loss after 30 min. pH 8.5 25 3. Percent loss after 15 min. pH 9.5 22 4. Percent loss after 30 min. pH 9.5 36 5. Percent loss after 15 min. pH 10.5 28 6. Percent loss after 30 min. pH 10.5 62

l Since the enzyme was very unstable at high temp. (70 C.), the combined eiect of high temperature and high pH was not performed.

i Not performed for same reason as stated for novo amylase.

EXAMPLE 10 To illustrate the ability of the ve microorganisms to produce alpha amylase in other media, a culture medium was prepared to the following composition:

Water, 1000 ml.

milliliter batches of the above-mentioned medium were placed in a 500 ml. baiiied shaking iiask and sterilized by steam in the ilasks at 121 C. for 15 minutes. Bacillus cereus A.T.C.C. No. 21768 through 21772 were independently inoculated into these fermentation media and cultivated under aerobic conditions at 37 C. for 48 hours at 250 to 900 rev./min. impeller speed which irriparts about 40 to 60 liters/min. air. The pH of the system varies from 6.0 to 9.0.

The microbial paste of the cells is stored at 20 C. The paste is then ruptured using a bacterial homogenizer at a temperature of 5 C. and .O1 ml. tris buffer, l0 liters EDTA (3 g.) and mercaptoethanol (8 ml.) are added. The protein containing supernatant is then fractionated with various polyethylene glycol concentrations of 50, 5, 10, 15, 20 percent. Each fractionation is allowed to stand at least 1 hour before centrifugation. The amount of alpha amylase accumulated in each cultured broth was as shown in Table 6.

TABLE 6 Conc., Carbon source percent 21768 21770 21771 21772 Wheat chat 2 73 40 60 39 Wheat bean- 2 62 42 60 44 Cracked cereal gra 2 51 40 37 39 Corn meal 2 40 30 37 38 Oatmeal 2 25 10 9 1l Rye meal 2 18 23 8 19 Cracked rice 2 15 31 5 23 Corn steep liquo 2 60 27 20 Starch (potato). 2 39 11 10 3 Wheat bram.-. 2 59 28 10 19 Soy bean oil 2 18 31 26 26 Soy beau meal.- 2 18 l5 10 1l Zein 2 19 15 10 11 Cotton seed inca 2 40 10 50 4l Casein 2 16 31 8 19 Wheat chaff 4 192 93 40 44 Wheat bran 4 65 90 43 50 Cracked cereal grain 4 64 113 33 39 orn me 4 32 20 31 40 Oatmeal. 4 26 28 11 16 Rye meal. 4 6 29 9 26 Cracked rice 4 11 30 11 23 Corn steep liquor.. 4 24 43 20 21 Starch (potato) 4 44 39 19 30 heat bran..." 4 23 44 11 16 Soy bean oil-. 4 11 26 l1 16 n 4 14 11 14 50 Cotton seed meal. 4 41 39 50 23 Cas D 4 17 15 11 23 Wheat cha. 6 190 88 43 60 Wheat bram 6 73 86 43 61 Cracked cereal gram.. 6 50 100 38 54 Com mea 6 30 23 31 30 Oatmeal 6 30 30 12 19 Rye meal 6 18 34 12 11 Cracked rl 6 37 11 19 Corn steep liquor.- 6 36 29 21 Starch (potat) 6 24 40 19 29 Wheat bran... 6 73 33 11 30 Soy bean oil.. G 26 10 Soy bean meal 6 11 Wheat chat! 10 40 85 5 30 Wheat bran 10 40 80 3 33 Cracked cereal grain 10 40 100 3 18 Corn meal 10 15 10 1 21 The following examples further illustrate the teachings of this invention with respect to variations in nutrient media and fermentation conditions.

EXAMPLE 11 About 500 m1. chicken waste culture medium were'prepared with the following composition:

Percent Chicken waste hulls, 80 mesh 5 (NH4)2HPO4 0.5 CaCO3 2.0 K2HPO4 0.1 CaClz 1 MgCl2 0.01

The medium was sterilized, inoculated with Bacillus cereus A.T.C.C. 21770 and cultivated with shaking at 37 C. for 60 hours. The culture broth so obtained was transferred 10% by volume) to a main culture medium of the composition given above which was cultivated with shaking at 37 C. A 50% urea solution was added during fermentation as needed to maintain the pH between 6 and 8, and chicken waste was supplied two times at a rate of 2% to make up for consumption. After 60 hours of fermentation, the concentration of alpha amylase in the culture medium amounted to 45 DNS units. This same experiment is repeated using Corn steep liquor rather than chicken waste and only 26-31 DNS units are produced.

EXAMPLE 12 About 500 ml. of chicken waste culture medium were sterilized in a 500 ml. Erlenmeyer batlled ask, the medium having the following composition:

Bacillus cereus A.T.C.C. 21768 was inoculated into the medium and cultivated at 30 C. for 18 hours, with shaking. During the fermentation, the pH was kept at 6 to 9 with a 50% urea solution. At the end of the cultivation period, alpha amylase accumulated in the culture broth amounted to 1,000 units/m1. or about 3.5 with the dnitrosalicylic acid test. Identical tests using Corn steep liquor rather than chicken waste produced 1.0 DNS units.

EXAMPLE 13 A culture brothof Bacillus cereus A.T.C.C. 21772 was cultivated in a culture medium composed of turkey waste at 30 C. for 18 hours and was inoculated into a main culture medium whose composition was:

Blackstrap molasses percent-- 1.0 (NH4)2SO4 ..-d0- 1.0 KH2PO4 do 0.2 MgSO47H2O do 0.05 Fe++ p p.m 2 Mn++ p.p.m 2 CaCO percent-- 1.5

The medium was held at 30 C., and 2% turkey Waste was added after 10, 24 and 48 hours. After 72 hours of cultivation, the 'amount of alpha amylase accumulated in theAcnlture broth was 80,."DNS1units. Repetition of the same experiment using Corn steep liquor in place of the turkey excrement produced 1.3 DNS units. Other carbon sources selected from Table 6 showed similar results.

EXAMPLE 14 500 ml. of seed culture broth of Bacillus cereus A.T.C.C. 21771 in a medium consisting of: (NH4)2HPO40.5%,l KH2PO40.2%, MgSO47H20 0.05% and yeast extract 0.05% is held at 30 C. for 20 hours with shaking and is inoculated into 200 ml. of a main culture medium in a 1000 ml. flask. The mixture is cultivated at 30 C. with shaking. The medium is composed added. At the end of a 48-hour fermentation period, the alpha amylase content of the broth was l DNS unit ml. A similar experiment using Corn steepliquor rather than chicken waste was made and resulted in the production of 0 DNS units of alpha amylase. Soy bean oil and wheat chaff substitutions produce similar data.

EXAMPLE 15 Bacillus cereus 21769 was cultivated in a medium consisting of: Yeast extract 0.37, malt extract 0.3% and soy bean oil -1%, pH 6, and further cultivated in the same culture medium and by the same method as in Example 14. At the end of the 48-hour fermentation period, the alpha amylase in the fermented broth amounted to 2 DNS/ml.

EXAMPLE 16 A starch culture medium was prepared and inoculated with Bacillus cereus 21770 and cultivated at 37 C. for

13 20 hours with shaking. The 300 ml. of the culture broth so obtained were inoculated into liters of a main culture medium composed of:

The medium was placed in a -liter fermentor and the fermentation was carried out at 30 C. with the air flowing at 10 liters/min. with stirring at 300 r.p.m. After 20 hours of fermentation, chicken waste was fed to the medium aseptically. Separately, 0.5% (NH4)2HPO4 was added and the pH maintained with aqueous ammonia solution. After 42 hours, 2 g./ml. 21 units valpha amylase was obtained in the broth.

EXAMPLE 17 Using Bacillus cereus 21771 the produceduredescribed in Example 16 was repeated. After 8 hours, 0.03% glucose was added; and at the end of the 48-hour fermentation, the concentration of alpha amylase in the fermented broth reached 81 DNS units.

EXAMPLE 18 Bacillus cereus A.T.C.C. No. 21770 was cultured in a medium containing bouillon 1%, NaCl 0.5% and yeast extract 0.1%, at 30 C. for 18 hours; and further in a main culture medium following the procedures of Example 13, at 30 C. for 72 hours. An addition of 0.01% potassium citrate was made after 12 hours of cultivation and 1.0% chicken waste was added after 12, 20, 30 and 40 hours. At the end of 72 hours, 36 DNS -units alpha amylase was obtained in the broth. Repetition of the experiment using various carbon sources takenv from Table 6 produce about 8-14 DNS units.

EXAMPLE 19 Aspergillus niger A.T.C.C. No. 15475 was inoculated into a medium of:

KHZPO., percentf 0.1 MgsojHzo perent "o.o5 (NH4)2SO4 pcrcent 0.1'- Urea percent-- 0.25 MnClz p.p.m 0.2 FeCl2 p.p.m 0.2

Chicken waste 120 mesh percent 2.0

The broth obtained after 20 hours at 30 C. was mixed with a main culture medium composed of:

After 18 hours of cultivation at 28 C. with shaking, the medium was adjusted to pH 7 to 8 with urea solution. After 12, 18, 24, 36 and 48 hours, 2% ethanol were added. At the end of 62 hours, the amount of alpha amylase in the fermented broth reached 1.3 units.

1 4 EXAMPLE 20 Bacillus cereus A.T.C.C. No. 21768 was inoculated in a culture medium consisting of:

Percent Nitrogen source, NaNo3 0.6 Chicken waste, hulls mesh 5.0 CaCO3 2 Acetate buffer, pH 6.8 CaCl2 0.1

The broth obtained after 24 hours at 37 C. was mixed with a main culture medium comprising:

Percent Chicken waste, 120 mesh 2.0 Acetic acid 1.0 (NH4)2SO4 0.5 Urea 0.5 KHPO4 0.2 K2HP04 0.2 MgSO47H2O 0.05 Aji-eki 1.5

,After 72 hours, the concentration of alpha amylase amounted to 29 units.

Fowl excrement utilization and analysis According to the teachings of this invention fowl excrement can serve as: 1) the original source for isolating the new alpha amylase producing microorganisms of this invention, (2) the sole ingredient in nutrient media for the new alpha amylase producing organisms of this invention as well as the s'ole nutrient for previously known alpha amylase producing microorganisms selected from the groups hereinafter described, and (3) a supplement to previously known nutrient media which can be used in the fermentation of both the new microorganisms of this invention and many previously known microorganisms hereinafter described.

The isolation and identification of this inventions new alpha amylase producing microorganisms from wet (nonsterile) fowl excrement sources is described in Example 2 of this application. The methods for obtaining the dried (sterile) fowl excrement used in the nutrient aspects of this invention are disclosed in assignees pending U.S. patent application Ser. No. 80,517 tiled Oct. 7, 1970. This application discloses suitable methods of collecting, sterilizing, drying, grinding and fractionating fowl excrement, particularly that obtained from commercial chicken and turkey farms. This patent application discloses that fowl excrement has a typical analysis of:

Ingredient: Percent by weight Crudefats 2.4 Silicates 1 .6 Carbohydrates 8.9 Phosphorus 2.0 Calcium 10.7 Magnesiumv 0.4 -Uric acid 0-5.0 Nitrogen 4.6 Crude fibers 11.8 Water 5.0 Remainder (inorganic anions and other carbon, hydrogen and oxygen moieties) 47.6

This analysis makes it evident that a high percentage of fowl excrement contains sources of assimilable carbon, assimilable nitrogen and minor nutrients. Proteinaceous material is clearly available in the form of the crude fats, carbohydrates, and crude fibers. The above chemical analysis clearly implies that all fractions of fowl excrement will contain nutrient qualities, but the applicants have further discovered that particularly high alpha amylase yields are obtained when the particle size of the fowl excrement is of a size greater than 350 mesh. The applicants data shows that it is even more preferable to use 15 fowl excrement of a size greater than 120 mesh and most preferable to use as much fowl excrement as possible of a size of greater than 80 mesh. Applicants have found this correlation to be true up to sizes of l mesh. Although the applicants do not wish to be bound to any theory to explain these differentials, they believe that they can be explained by the fact that the larger fractions of fowl excrement tend to have higher carbohydrate con- The media employed had the following compositions.

Media Size Media tents.

A. Chicken excrement i 1 0 EXAMPLE 21 B-.. o 120 mesh i2 0 C Turkey excrcment do l 2. 0 Bacteria and yeasts of other known and widely varying Chlfken exCrement-u 12.0 genera are capable of producing alpha amylase on media 0 l 121g whose sole or principal source of assimilable carbon and 12.0 assimilable nitrogen is fowl excrement. The strains of ,gz microorganisms suitable for producing alpha amylase do 01 .not share readily recognizable features other than their p O' ability to grow on la culture medium containing fowl exhilen excremntiso Cremeut as the principal source of assimilable carbon and Cgchfj "j (2):? nitrogen and producing alpha amylase by metabolizing liglgi- :0.1 this medium. Employing screening tests based on these pLf.: all; properties, we have found numerous suitable micro- I increment i 3.0 organisms in nature and in culture collections without e o o :3 '1g exhausting the field of search. 02 5 Table 7 below lists the new strains of this invention 25 11(2) along with many previously known microorganisms, as I Ch k m nt 11,000 well as their growing ability on a fowl excrement. media. Cebgsffffnj 33(1) This growth was conrmed in the following manner. Mfsoi-JHZO. 1 10 Plate cultures of the microorganisms were incubated for Cacggiig" ,0 3 5 days at 30 C. and a piece of the liquid contents were 30 geast rafhr :1.0 transferred to lter paper by a diffusion technique. The Dgled 21 'gbg alpha amylase Content 0f each Culture medium was cal- K 'ulrlkgy excrement- :5,0 culated by the dinitrosalicylic acid procedureas well as Msbfsgzo" :01(1) other methods of enzymology. Chromotographic data was K 2 5 used to determine what sugars were produced. The growth 2:3 of microorganisms is determined by plate counts and gillsrtliiied 1110....t 11,000 utilization of sugar, DNA and RNA. The results are eval- L Cellogfogcfff; 13:2 uated in Table 7 on an arbitrary, but reproducible scale, MgJSot-.YHL io the symbols used having the following meaning. stzL- gg-)t ex .0 -l-l--l--i- EXCelleDt 40 Distilled H20 l 1,000 -l--l--l- Good Fair 1 Percent of medafby weight.

I l Grams per liter. -l- Signicant comms. Trace 4 Minuit/eis.

TABLE 7 Nutrient media A B C D E F G H I K L Aspergillus zu/amori Too 11358 -l- -I- Awefgllw diallws -l- -l--l- Aspergillus flaws ATCC 1149 -l- -l- -l--I- -l- Aspergillus foetidus ATCC 14916 -l--l- Aspergillus niger ATCC 15475.. 'i' Aspergillus ori/:ae ATCC 7561..- +l+ l+ Aspergillus ori/zae ATCC 9102 -l- -l--l- Aspergillus phoencis ATCC 15556. Aspergillus ruber ATCC 9481 Aspergillus saitol ATCC 11362. 'i- *i* Aspergillus terreus ATCC 11877 Aspergillus usamii ATCC 11364 Bacillus amyloliquefaciens ATCC 23843.. -r'l' 'i' Bacillus brenis ATCC 10068 -i- Bacillus cereus ATCC 944.. -l- -l- Bacillus cereus ATCC 7004. -l- -l--i- Bacillus cereus ATCC 12480 Bacillus cereus ATCC 21768.. -l-'i ll -l Bacillus cereus ATCC 21769.... -l--lnl i 'l -i- Bacillus cereus ATCC 21710.... lri- +4 Bacillus cereus ATCC 2l771 -l--l--l- -r|+' Bacillus cereus ATCC 21772.... -l--I- -l--i--l- Candida salami ATCC 14440- -l- Candida parapsolosis Cellumonas biazotea ATCC 486. -l- Cellumonas fum' ATCC i5724 Cargnebacteria lilium ATCC 15090-. Corynebacteria hydrocarbaclastus 1955 Pseudomonas aerginasa ATCC 7706- Pseudomonas amyloderumosa ATCC Pseudomonas atrofaceus ATCC 9004.. -l--l- Fermentations are again carried out by employing microorganisms listed in Table 7 in the same nutrient media and under the same conditions as those described in Ex ample 21 except that the fowl excrement percentages are Fermentations were carried out by employing each of the microorgansm listed in Table 7 vunder the same fermentation condition listed in Example 21 media .l except changed as indicated in Table 8. The fowl excrement is 5 the fowl excrement sized to 120 mesh fwas added to the sized to 350 mesh. The medium employed has a carbon nutrient media in the amounts shown 1n Table 8. This source of 7.0% corn steep liquor. The amylase produccomparison shows the beriecial results of using the larger fractions of fowl excrement.

Alpha amylase produced 0. 0% 0. 1% 2% 4% 6% 8% 10% 20% 4% 5% 8% Mlcroorgunism employed CW CW CW CW CW CW CW CW TW TW TW Aspergillus diastalicus 0 0 1. 7 1. 9 2.1 0 0 0 1. 7 l. 9 0 Aspergillusjluvus ATCC 114 0 0 1.5 1.6 2.0 1.1; 0 0 1.5 1.0 0 Aspergillus faezidm ATCC 1491s o.A a 1. 0 1. 0 o o o o o ov 0 Aspergillus niger ATCC 15475- o o 1.3 1. 0 o 0v o 0 1. i 1.3 o Azpergillus orgzae ATCC 7561 0 0 1. 0 1. 1 1. 1 7 0 0 0 0 0 Aspergillus org/:ae ATCC 9102 0 0 1. 1 1. 5 1. 0 0 0 0 0 0 0 Aspergillus phocnicis ATCC 0 0 O 1. 0 l. l 0 0 0 0 0 0 Aspergillus ruber ATCC 9481. 0 0 0 0 0 0 0 0 1. 7 2. 1 3. 0 Aspergillus saitol ATCC 11362 0 0 0 0 0 0 0 0 1.0 0 0 Aspergillus usami ATCC 11364 0 0 0 0 0 0 0 0 1.5 1.9 2. 3 Bacillus amylolique faccru ATCC 23843. 0. 5 0. 5 1. 1 1. 3 1. 3 0 0 0 1. 1 1. 4 1. 4 Bacillus brevis ATCC 10068 0. 1 0. 1 1. 3 1.5 1.5 0 0 0 1. 2 1. 4 1. 4 Bacillus cereus ATCC 21768 0.2 2. 3l 9 38 9 78 0 0 78 80 96 0. 2 2. 3 2. s 46 71 76 o o 78 so 93 0.3 2.5 2.7 42 s3- Vero 0 s3 se 91 0. 2 2. 3 2. 7 45 71 60 (lv 0 71 75 0 0. 2 2. 7A 2. 8 49 11 63 0 0 71 75 0 6.0 0.5 3 9 0 0 0 0 0 0 0 .900 v 1. 1 4 3 0 0 0 0 0 0 0 0 1. 0 6 5 0 0 0 0 0 0 0 Cellumonas cartali/tcum ATCC 21 0 0.5 1 1 0 G 0 0 0 0 0 Cellu'mo'nas jlavlgena ATCC 4212..... 0 0. 5 6 1 0 0 0 0 0 0 0 Pseudomonas aerginom ATCC 7706 0 0` 0 0 0 0 0 0 1.5 2 9 Norn.-CW==chlcken waste; TW=turkey waste.

tion of these various microorganisms expressed in DNS` units is given in Table 8 below.

TABLE s 4vSirriilai""excl:rements are carried out using media A, C, D, G, K and L with similar results being obtained.

j Alpha amylase produced l 0. 0% 0. 1% 2% 4% 6% 8 10% 20% 4% 5 a 8% Microorganlsm employed CW CW CW CW CW C CW CW TW Ta' TW Aspergillus dataticus 0 1 1-1 2.0'. 2.1 1.6 0 0 2.7 3.1 2.1 1' Aspergillus jlavua ATCC 11495.- 0 1.3 1.6- 2.4. 1.5 0 0 0 1.7 1. 9 1.3 Aspergillus foetidus ATCC 14916. 0 3. 0 1. 8` 2. 4 1. 9 0 0 0 1. 7 1. 9 1. 0 Aspergillus 'niger ATCC 15475... 0 0 .5 1. 3 1. 1 0 0 0 1. 1 1. 8 1. 1 Aspergillus er1/:ue ATCC 7561. 0 0 0 0 1.0 0 0 0 3. 9 4. 1 8.6 Aspergillus ors/zas ATCC 9102... 0 0 1.4 1.9 1.0 0 0 0 3.0 3.8 8.0 Aspergillus phoenicis ATCC 15556- 0 l 0 0 0v 0 0 0 0 1. 1 1. 3 1. 1 Aspergillus usamii ATCC 11364..... 0 0 0 0 0 l 0 0 0 2. 9 3. 1 3. U Bacillus amyloliquefaciens ATCC 2384 0.5 1. 4 1 1.8' 2. 1 1.5 1.0 0 0 1.5 1.9 1.4 Bacillus bravia ATCC 10068 0. 5 1. 1 1. 4 2. 0' 1. 0 0 0 0 1. 0 1. 0 1. 0 Bacillus cereus ATCC 12480. 0. 5 0 (1 0 0 0 0 0 1. 1 1. 6 1. 1 Bacillus cereus ATCC 21768. 0.5 3 7 28 38 12 0 0 29 36 29' Bacillus cereus ATCC 21769. 0. 1 7 14 32V 38 24 17 0 30 38 21 Bacillus cereus ATCC 21770. 0 9 15 32' 30 0 0 0 33 39 25 Bacillus cereus ATCC 21771. 0 9 11 40 46 0 21 0 30 37 29 Bacillus cereus ATCC 21772. 0. 1 11 18 43 48 0 0 0 0 U 0 Candida salami ATCC 14440.- 0 0 0 1.0 0 l) 0 0 0 0 0 Candida parapsolosis ATCC 0 0 0 1. 1 0 0 0 0 0 0 Candida guillier mondii ATCC 9390. 0 0 0 1. 1 0 0 0 0 0 0 0 Cellumonus biazotca ATCC 486.-.. 0 0 1. 1 1. 0 0 0 0 0 0 0 0 Cellumonas 'mi ATCC 15724 0 0 1.3 2. 0 0 0 0 0 0 0 0 Corynebacleria M/drocarboclaslus ATCC 195 0 0 0 1. 1 0 0 0 0 0 0 0 Pseudomonas amyloderamosa ATCC 21262 0 0 0 1. 1 0 0 0 0 0 0 0 Pseudomonas atrofaciem ATCC 9004 0 (lv 0 1. 1 0 0 0 0 0 0 0 EXAMPLE 24 Similar excrements are carried out using media A, C, G and K of Example 2l and similar results are obtained.

Fermentations are again carried out using the conditions of Example 23 media I except that the fowl excrement was of size 80 mesh or greater. The results indicate that alpha amylase production was improved with the 75 larger fractions.

Alpha amylase produced CW CW CW CW TW TW TW CW CW CW CW Microrgsnism employed .NQT;:.-CW @maken waste; TW=urkey waste.

2l EXAMPLE 27 Fermentations were carried out by employing microorganisms listed in Table 7 under the same conditions as 1n Example 2l media L except that the carbon source is extract, soybean meal as well as mixtures of the above. We prefer to employ about 0.8% proteinaceous material in our nutrient media as this amount helps to insure good yields of amylase.

10% wheat chai. 5 As assimilable carbon sources, our process can utilize Alpha amylase produced 0. 0% 0. 1% 2% 4% 6% 8% 10% 20 4 5 B Microorganism employed CW CW CW CW C W CW CW Clnl T158 T1171'l Tg5 Aspergillus dastatcus 0 1. 5 3. 7 21 43 28 0 0 31 34 B Aspergillus jiavus ATCC 11495. O 1. 2 4. 4 21 43 30 0 0 28 31 11 Aspergillus foetidus ATCC 1491 0 1. 3 4. O 21 46 28 0 0 15 18 3 Aspergillus niger ATCC 15475 0 1. 9 4. 3 20 45 30 0 0 31 34 6 Aspergillus oryzae ATCC 7561 0 1. 7 4. 6 0 0 0 0 0 0 0 0 Aspergillus orgzae ATCC 9102. 0 1. 3 4. 2 21 43 31 0 0 0 0 0 Aspergillus phoem'cis ATCC 15556. 0 1. 3 4. 1 21 45 28 O 0 0 0 0 Aspergillus ruber ATC C 9481.- 0 1. 4 4. 1 22 43 31 0 0 0 0 0 Aspergillus saitol ATCC 11362- 0 1.3 4. 0 21 45 28 0 0 35 39 31 Aspergillus usamii ATCC 11364 0 1.3 3. 6 9 19 0 0 0 0 0 0 Bacillus amgloliquijaciem ATCC 23843. 0 3. 1 11 20 17 0 0 0 26 28 16 Bacillus brevis ATCC 10068 0 2. 6 21 21 17 0 0 0 28 31 11 Bacillus subtilis 0 0 0 0 0 0 0 0 9 11 9 Bacillus cereus ATCC 21768- 0 3. 0 9. 0 110 89 96 21 0 93 101 108 Bacillus cereus ATCC 21769 0 8. 1 7. 4 92 99 30 0 96 100 106 Bacillus cereus ATCC 21770.. 0 6. 3 7. 2 96 103 50 0 96 102 107 Bacillus cereus ATCC 21771-- 0 7. 0 7. 1 92 140 32 0 93 101 0 Bacillus cereus ATCC 21772 0 7. 3 23 100 142 32 0 95 101 0 Cellumouas bazotea ATCC 486 0 14 28 36 16 0 0 0 0 0 Cellumonas )imi ATCC 15724 0 15 30 38 10 0 0 0 0 0 Cellumona.: vibroides ATCC 11764 0 17 33 36 11 0 0 0 0 0 Cell v carff'l ATCC 21621 0 14 31 34 16 0 0 0 0 0 Cellumonas jlavige-na ATCC 482 0 11 30 38 10 0 0 0 0 0 N0'rE.-CW=chicken Waste; TW=turkey waste.

EXAMPLE 28 glucose, arabinose, xylose, lactose, maltose, sucrose, etc.

Fermentations are again carried out by the microor- 35 and various organic compounds, such as organic ammonium salts, organic nitrate, urea amino acids, corn steep liquor, peptone, casein, meat extracts, or soy products, etc. These materials of course can be used not only as a carbon source but also as digestible nitrogen source. Ob-

40 viously we also use the uric acid in the chicken waste or Asperiglus ATCC ATCC ATCC ATCC ATCC nig., TCC

Bacillus cereus 21768 21769 21770 21771 21772 15475 10% wheat chaff plus glycine 330 203 85 5 3D 1.0

5% wheat cha plus glycine 130 17 89 31 39 1. 1

EXAMPLE 29 turkey waste as a source of nitrogen. Other sources of ni- Repeat Example 28 using 5% and 2% chicken waste media. Results:

trogen for this invention can be any organic or inorganic nitrogen-containing compound which is capable of releas- Aspergillus ATCC ATCC ATCC ATCC ATCC nig., ATCC Bacillus cereus 21768 21769 21770 21771 21772 15475 5% CW plus 200 pg. glycine 135 33. 5D 94 42 29 2. 85 3% CW plus 200 ng. glycine 119 23. 35 94 44 47 2. 80

According to a particularly preferred embodiment of this invention fowl excrement can be employed as a supplement to those nutrient media which are used in conventional alpha amylase fermentation processes. This point is Well illustrated in the examples of this application. However, the amounts of fowl excrement empolyed as a supplement in conventional nutrient media may vary over a relatively wide range. Concentrations as low as 0.01 and as high as 15 percent by weight of the conventional media are effective to increase the yield of alpha amylase. The ap` plicants have discovered that concentrations in excess of 15 percent by weight confer no added advantages and in some cases concentrations above 5% act as inhibitors. The preferred concentration is therefore in the range of 1 to 5 percent and the most preferred is 2-5 percent by weight of the conventional media.

In using fowl excrement as a supplement to conventional nutrient media the applicants prefer to prepare a nutrient medium containing between 0.01 and 15% proteinaceous material and starch in amounts between 0.01 and 15%, the medium being made up to volume with Water. Proteinaceous material suitable for use in the practice of our invention includes corn steep liquor, casein, dry yeast, sh meal, cottonseed meal, yeast extract, meat ing nitrogen in a form suitable for metabolic utilization by the microorganism(s) being harvested. In the organic category, the following compounds can be listed as exemplary nitrogen-containing compounds which can be used: proteins, acid-hydrolized proteins, enzymedigested proteins, amino acids, yeast extract, asparagine, urea, uric acid etc. and it is obvious that such materials can also be utilized as carbon sources. For reasons of eco nomy, it is ususally preferable to empoly inorganic nitrogen compounds, such as ammonia, ammonium hydroxide, or salts thereof, such as ammonium citrate, ammonium sulfate, ammonium phosphate, ammonium acid phosphate. As noted earlier, turkey and chicken waste can be used as l the sole source of carbon and nitrogen. Another very convenient and satisfactory method of supplying nitrogen is to employ ammonium phosphate or ammonium acid phosphate, which can be added as the salt, per se, or can be produced in situ in the aqueous fermentation media by bubbling nascent nitrogen through the broth to which phosphoric acid was previously added, thereby forming ammonium acid phosphate.

Starch source which we can empoly in our nutrient media can be either soluble starches, or any of the common insoluble starches such as rice starch, corn starch,

23 potato starch, arrow root starch, etc. The source of the starch is not considered to be critical and consequently, we intend to include in the term starch all of the various known forms, whatever their source. The role of starch is in the nature of a template or stimulant and the amount to be used falls within a range of about 0.01 to about We have obtained particularily satisfactory results employing 0.5% starch and therefore prefer this amount.

In addition to the carbon and nitrogen sources, it may be necessary with some organisms to supply requisite amounts of selected mineral nutrients in the nutrient medium in order to insure proper microorganism growth and maximize selectivity, viz, the conversion of fowl excrement and other nutrients to microorganism cells. Thus, potassium, sodium, iron, magnesium, calcium, manganese, phosphorous, and other nutrients can be included in the aqueous growth medium. However in most cases chicken waste and turkey waste contain enough minerals for growth and production of alpha amylase. In any event, these -materials can be supplied in form of their salts, and preferably their water-soluble salts. For example, the potassium can be supplied as potassium chloride, phosphate sulfate, citrate, acetate, nitrate, etc. Iron and phosphorous can be supplied in the form of sulfates and phosphates, respectively, e.g., iron sulfate, iron phosphate. Usually most of the phosphorous is supplied as ammonia phosphate. When either ammonium phosphate or ammonium acid phosphate is used, it can serve as a combined source of both nitrogen and phosphorous (phosphate ion) for microorganism cell growth.

Following inoculation, the fermentations of this invention are conducted in a temperature ranging of about C. to about 50 C. within which limits optimum growth occurs. However, we prefer to maintain the temperature in the range 26 to 37 C. and more particularly 32-35 C., at which temperature we have obtained the best results. During the fermentation, we maintain the pH of the medium at a value ranging between about 3.5 and 9.5 within which range the organism grows satisfactorily. However we prefer to employ a pH of about 7.2 at which we have obtained the best results.

The above organisms are aerobic and thus must be grown in the presence of air if adequate growth is to occur. Consequently, the organism can be grown on the surface of shallow layers of the medium or it can be grown in aerated submerged cultures, wherein deep tanks are employed with air being supplied thereto. We have employed both processes and found them satisfactory in the production of amylase, the deep tank method of course being the most satisfactory insofar as commercial production is concerned.

The fermentation is continued until the maximum yield of alpha amylase is obtained. Generally the time of the fermentation can be said to range from 12 to 72 hours alalthough we have demonstrated that amylase activity is present in the medium in as short a period as four hours from the time the fermentation is begun. Generally the yield reaches a maximum after about 12-16 hours but considerably less than the longer time limit of 72 hours and consequently we prefer to carry out the fermentation for a period of about 30 hours.

Oxygen can be supplied to the cultivation medium in any form capable of being as similated readily by the inoculant microorganism, and oxygen-containing compounds can be used as long as they do not adversely affect microorganism cell growth. However, oxygen is conveniently supplied as an oxygen-containing gas, e.g., air, which contains from 19 to 22 wt. percent oxygen. While it is preferable to employ air, oxygen enriched air having more than 22 wt. percent oxygen e.g., enriched air having in excess of 22 wt. percent oxygen, can be used.

Following recovery of the precipitated amylase from the medium, the material is dried to obtain the final preparation having exceptional alpha amylase activity. The

drying can be effected in vacuo at room temperature over calcium chloride or it can be effected at temperatures up to about 65 C. without decrease in the activity of the alpha amylase preparation.

Following production of the amylase in the nutrient medium, we then recover the amylase by first removing the solids from the medium and then precipitating the amylase from the cell-free medium by the addition of a precipitating agent more particularly described below. The solids can be removed from the medium in any of the usual ways such as for example by filtration, centrifugation, etc. Many times it is advantageous to employ the alpha amylase in solution and consequently in such situations precipitation is not necessary, the solution remaining following removal of the cells from the nutrient medium being satisfactory for this purpose. However concentration of the cell-free media improves their potency and such procedure is preferred.

After separating the solids from the nutrient medium and obtaining the cell-free solution of the amylase, we prefer to precipitate the alpha amylase by adding to the medium precipitating agents such as organic water-miscible solvents. Examples of such precipitation agents are ammonium sulfate, methyl ethyl ketone, acetone, dioxane, and lower aliphatic alcohols including methanol, ethanol, isopropanol, n-propyl alcohol, tertiary butyl alcohol, polyethylene, or mixtures thereof. We add enough of the solvent to precipitate all of the amylase but generally we have found that from about 0.5 to 2.0 volumes of the precipitating agent per volume of cell-free medium are sufficient to precipitate all of the alpha amylase. Generally we prefer to employ ammonium sulfate as the precipitating agent since it appears to give equally good results no matter what the concentration of amylase in the cell-free nutrient medium might be. We prefer to employ 2 volumes of polyethylene glycol per volume of cell-free nutrient medium containing the amylase since we are generally assured of complete precipitation when this amount is employed.

In connection with the precipitation of the alpha amylase, we have found it desirable to concentrate the cellfree nutrient medium prior to effecting the precipitation since such procedure decreases the amount of precipitating agent needed to obtain a given amount of amylase. We prefer to concentrate the solution to about one-tenth its original volume. Similarly, we have found it desirable to dialyze the nutrient medium prior to concentration since such procedure increases the purity of the final product. We prefer to dialyze the solution in cellophane bags against running tap water for a period of about 24 hours when such procedure is not impractical.

At the start-up of the fermentation the sterile growth medium is inoculated with the microorganism to be harvested, e.g., by use of previously cultivated inoculum in a growth media or the same one in which it is to be grown for production, e.g., as described above. The initial concentration of inoculum containing said microorganism at the outset of fermentation can vary widely, e.g., 0.0005 to 50.0 grams per liter of total fermentation media. Other inoculation procedures can be employed, e.g., use of an inoculum where said microorganism is previously grown on a media different from that in which the fermentation is to be conducted and then transferred to the fermentation vessel(s) etc. Normally the culture is grown for 14-18 hours before being inoculated in the production media.

Having thus disclosed our invention, we claim:

1. In a process for producing alpha amylase by fermenting under aerobic conditions, by means of microorganisms, a nutrient medium containing a source of starch, proteinaceous material and mineral salts, the improvement comprising using 0.01 to l0 percent by weight of dried fowl excrement sized between l0 and 350 mesh in said nutrient, medium, and wherein said microorganisms are selected from the group consisting of those Pseudomonas, Aspergillus, Cornyebacteria, Cellumonas, Bacillus and Candida capable of producing alpha amylase by metabolizing fowl excrement.

2. The process of claim 1 wherein the Bacillus are selected from the group consisting of Bacillus brevis, Bacillus amyloliquefaciens, and Bacillus cereus.

3. The process of claim 1 wherein the Bacillus cereus are selected from the group consisting of Bacillus cereus 21768, Bacillus cereus ATIC No. 21769; Bacillus cereus No. 21770; Bacillus cereus ATCC N0. 21771 and Bacillus cereus No. 21772.

4. The process of claim 1 wherein the Pseudomonas are selected from the group consisting of: Pseudomonas aeruginosa, Pseudomonas amyloderamosa and Pseudomonas atrofaciens.

5. The process of claim 1 wherein the Aspergillus are selected from the group consisting of: Aspergillus awamori, Aspergillus diastaticus, Aspergillus flavus, Aspergillus foetidus, Aspergillus niger, Aspergillus orzae, Aspergillus phoenicis, Aspergillus ruber, Aspergillus saitol, Aspergillus terras and Aspergillus usamii.

6. The process of claim 1 wherein the Corynebacteria are selected from the group consisting of Corynbacteria illiwn and Corynebacteria hydrocarboclastus.

7. The process of claim 1 wherein the Cellumonas are selected from the group consisting of: Cellumonas biazotea and Cellumonas fimi.

8. The process of claim 1 wherein the Candida are selected from the group consisting of Candida salami and Candida parapsolosis.

9. The process of claim 1 wherein the fowl waste is in the size range of 120 to 50 mesh.

10. The process of claim 1 wherein the fowl waste is in the size range of 100 to 80 mesh.

11. The process of claim 1 wherein the nutrient medium comprises water, fowl excrement, and between 0.01 and percent starch and between 0.01 and 15 percent proteinaceous material.

12. The process of claim 1 wherein the mineral salts are selected from the group consisting of carbonates, phosphates, and sulfatos of iron, manganese, calcium, potassium, sodium and magnesium.

13. The process of claim 11 wherein the starch source is selected from the group consisting of rice starch, corn starch, potato starch, arrowroot starch, wheat starch, cassava starch.

14. The process of claim 11 wherein the protein source is selected from the group consisting of soya bean meal, cotton seed meal, corn steep liquor, zein, prosein, casein, corn meal, homing, oat meal, rye meal, wheat meal, wheat chai and broken rice.

15. The process of claim 1 wherein the fermentation is carried out in the temperature range of 18 to 70 C.

16. The process of claim 1 wherein the fermentation is carried out in the temperature range of 26 to 37 C.

17. The process of claim 1 wherein the fermentation is carried out in a pH range of 1 to 11.

18. The process of claim 1 wherein the fermentation is carried out in the pH range of 6.5 to 7.5.

19. The process of claim 1 wherein the fermentation is carried out for a period of 1 to 72 hours.

20. The process of claim 1 wherein the fermentation is carried out for a period of 25 to 35 hours.

21. The process of claim 1 wherein the fermentation is carried out in a nutrient medium containing growth promoting portions of wheat chai and glycine in conjoint presence in said nutrient media.

22. The process of claim 20 wherein the wheat chai is present in amounts of from 1 to 15% and the glycine is present in amounts of from 0.01 t0 5%.

References Cited UNITED STATES PATENTS 3,723,250 3/ 1973 Aunstrup 195-62 3,455,696 7/ 1969 Ukita et al 99-9 3,546,812 12/1970 Kobayashi et al. 99--9` X 3,633,547 1/ 1972 Stevens et al.

LIONEL M. SHAPIRO, Examiner U.S. Cl. X.R. --28 R 

